Fallopia japonica Root Extract Ameliorates Ovalbumin-Induced Airway Inflammation in a CARAS Mouse Model by Modulating the IL-33/TSLP/NF-κB Signaling Pathway

Fallopia japonica (Asian knotweed) is a medicinal herb traditionally used to treat inflammation, among other conditions. However, the effects of F. japonica root extract (FJE) on airway inflammation associated with combined allergic rhinitis and asthma (CARAS) and the related mechanisms have not been investigated. This study examined the effect of FJE against CARAS in an ovalbumin (OVA)-induced CARAS mouse model. Six-week-old male BALB/c mice were randomly segregated into six groups. Mice were sensitized intraperitoneally with OVA on days 1, 8, and 15, and administered saline, Dexamethasone (1.5 mg/kg), or FJE (50, 100, or 200 mg/kg) once a day for 16 days. Nasal symptoms, inflammatory cells, OVA-specific immunoglobulins, cytokine production, mast cell activation, and nasal histopathology were assessed. Administration of FJE down-regulated OVA-specific IgE and up-regulated OVA-specific IgG2a in serum. FJE reduced the production of T helper (Th) type 2 cytokines, and the Th1 cytokine levels were enhanced in nasal and bronchoalveolar lavage fluid. Moreover, FJE positively regulated allergic responses by reducing the accumulation of inflammatory cells, improving nasal and lung histopathological characteristics, and inhibiting inflammation-associated cytokines. FJE positively modulated the IL-33/TSLP/NF-B signaling pathway, which is involved in regulating inflammatory cells, immunoglobulin levels, and pro-inflammatory cytokines at the molecular level.


Introduction
Allergic asthma is a chronic and common respiratory disease that is a major health concern globally, affecting individuals of all ages, and is estimated to impact 339 million people worldwide. Another 100 million people are anticipated to be diagnosed with asthma by 2025 [1]. Allergic rhinitis (AR) is an allergic-mediated inflammatory condition that causes nasal irritation, congestion, and sneezing and is a key risk factor for asthma [2]. If AR is managed poorly, the risk of asthma increases several fold [3]. Recently, combined allergic rhinitis and asthma syndrome (CARAS) has emerged as a novel disorder associated with lower and upper lung inflammation [4]. AR and asthma are closely associated, as they are triggered by similar etiological factors, exhibit similar symptoms, and respond to equivalent therapeutic interventions [4]. Understanding the pathogenesis of CARAS is crucial for developing potential treatments. The pathogenesis of CARAS involves complex

FJE Suppressed Rat Peritoneal Mast Cell (RPMC) Degranulation
To examine the influence of FJE on mast cells, which play a critical role in anti-allergic inflammatory responses, RPMC degranulation was assessed. Compound 48/80 (C48/80) leads to mediator exocytosis and RPMC degranulation by stimulating the signal transduction pathway. We observed that normal RPMCs had packed secretory granules, were oval, and showed a clear cell outline. RPMCs treated with FJE revealed characteristics similar to normal RPMCs, irrespective of the concentration used. However, RPMCs induced with C48/80 showed indistinct cell outlines, and the expression of intracytoplasmic granules on the cell surface indicated substantial degranulation ( Figure 1A). FJE supplementation at 0.1-10 mg/mL after stimulation with C48/80 suppressed the degranulation of mast cells ( Figure 1B).

FJE Inhibited Nasal Symptoms in the OVA-Induced CARAS Mouse Model
The frequencies of nasal rubbing and sneezing were recorded to examine the effects of FJE on the OVA-induced CARAS mice. Mice in the CARAS group revealed significantly higher rates of nasal rubbing and sneezing than those in the control group. Notably, FJE reduced the sneezing and rubbing incidences in a dose-dependent manner, with significantly lower numbers of events than in the CARAS group ( Figure 1C,D).

FJE Inhibited Nasal Symptoms in the OVA-Induced CARAS Mouse Model
The frequencies of nasal rubbing and sneezing were recorded to examine the effects of FJE on the OVA-induced CARAS mice. Mice in the CARAS group revealed significantly higher rates of nasal rubbing and sneezing than those in the control group. Notably, FJE reduced the sneezing and rubbing incidences in a dose-dependent manner, with significantly lower numbers of events than in the CARAS group ( Figure 1C,D).

FJE Reduced Infiltration of Inflammatory Cells in the Nasal Lavage Fluid (NLF) and BALF of the OVA-Induced CARAS Mouse Model
Activation of the inflammatory response plays an essential role in the development of CARAS. To assess the anti-inflammatory effects of FJE on CARAS, inflammatory cells were quantified in NLF and BALF. As shown in Figure 2A,D, the total number of inflammatory cells was significantly higher in the CARAS group than in other groups. Administration of FJE led to a dose-dependent reduction in inflammatory cells; groups that received FJE doses of 100 and 200 mg/kg had a significantly lower number of inflammatory cells than the CARAS group. Moreover, the CARAS group had greater numbers of epithelial cells, macrophages, eosinophils, neutrophils, and lymphocyte cells than other groups. The FJE-treated group demonstrated a dose-dependent reduction in the numbers of epithelial cells, macrophages, eosinophils, neutrophils, and lymphocytes ( Figure  2B,C,E,F). Collectively, these observations suggest that FJE suppressed the total number of inflammatory cells in the NLF and BALF. These observations indicate the positive influence of FJE on the airway inflammation of the CARAS mice. Data represent mean ± SEM (n = 6/group). ### p < 0.001 compared with control, ** p < 0.01, *** p < 0.001 vs. CARAS. Dex, Dexamethasone.

FJE Reduced Infiltration of Inflammatory Cells in the Nasal Lavage Fluid (NLF) and BALF of the OVA-Induced CARAS Mouse Model
Activation of the inflammatory response plays an essential role in the development of CARAS. To assess the anti-inflammatory effects of FJE on CARAS, inflammatory cells were quantified in NLF and BALF. As shown in Figure 2A,D, the total number of inflammatory cells was significantly higher in the CARAS group than in other groups. Administration of FJE led to a dose-dependent reduction in inflammatory cells; groups that received FJE doses of 100 and 200 mg/kg had a significantly lower number of inflammatory cells than the CARAS group. Moreover, the CARAS group had greater numbers of epithelial cells, macrophages, eosinophils, neutrophils, and lymphocyte cells than other groups. The FJEtreated group demonstrated a dose-dependent reduction in the numbers of epithelial cells, macrophages, eosinophils, neutrophils, and lymphocytes ( Figure 2B,C,E,F). Collectively, these observations suggest that FJE suppressed the total number of inflammatory cells in the NLF and BALF. These observations indicate the positive influence of FJE on the airway inflammation of the CARAS mice.

FJE Suppressed Histopathological Alterations in the Nasal Mucosa and Lung Tissue of the OVA-Induced CARAS Mouse Model
Nasal mucosal thickness was significantly greater in the CARAS group than in the other groups. Mice administered with FJE and Dexamethasone (Dex) showed less mucosal edema than CARAS mice. Moreover, the subepithelial layer of the nasal mucosa was significantly thinner in mice treated with 200 mg/kg of FJE and Dex than in the CARAS mice ( Figure 3A-C). Furthermore, the bronchus showed significant changes in the mice of the CARAS group compared with control mice ( Figure 3E). PAS staining showed less goblet cell hyperplasia in FJE-and Dex-treated mice than in the CARAS mice. Interestingly, OVA-activated goblet cells exhibited mucus hypersecretion in the nasal and lung tissues of CARAS mice. Dex and FJE (100 and 200 mg/kg) supplementation suppressed mucus overproduction ( Figure 3F). Additionally, toluidine blue staining of mast cells showed higher numbers in CARAS mice compared with FJE-treated mice ( Figure 3G), whereas, in the CARAS mice, there was a greater number of eosinophils. Eosinophils are stained red in the cytoplasm Red arrows indicate eosinophils. Mice were administered saline, Dex (1.5 mg/kg), or FJE (50, 100, or 200 mg/kg) once a day for 16 days. Data represent mean ± SEM (n = 6/group). ### p < 0.001 compared with control, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CARAS. Dex, Dexamethasone.

FJE Suppressed Histopathological Alterations in the Nasal Mucosa and Lung Tissue of the OVA-Induced CARAS Mouse Model
Nasal mucosal thickness was significantly greater in the CARAS group than in the other groups. Mice administered with FJE and Dexamethasone (Dex) showed less mucosal edema than CARAS mice. Moreover, the subepithelial layer of the nasal mucosa was significantly thinner in mice treated with 200 mg/kg of FJE and Dex than in the CARAS mice ( Figure 3A-C). Furthermore, the bronchus showed significant changes in the mice of the CARAS group compared with control mice ( Figure 3E). PAS staining showed less goblet cell hyperplasia in FJE-and Dex-treated mice than in the CARAS mice. Interestingly, OVA-activated goblet cells exhibited mucus hypersecretion in the nasal and lung tissues of CARAS mice. Dex and FJE (100 and 200 mg/kg) supplementation suppressed mucus overproduction ( Figure 3F). Additionally, toluidine blue staining of mast cells showed higher numbers in CARAS mice compared with FJE-treated mice ( Figure 3G), whereas, in the CARAS mice, there was a greater number of eosinophils. Eosinophils are stained red in the cytoplasm and marked with black head arrows in Figure 3H. These findings strongly indicate that FJE administration alleviated pathological injury in mice with OVAinduced CARAS.   Data represent mean ± SEM (n = 6/group). ### p < 0.001 compared with control, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CARAS. Dex, Dexamethasone.

FJE Suppressed Immunoglobulin Levels in Serum of the OVA-Induced CARAS Mouse Model
To investigate whether FJE affects allergic reactions, OVA-specific IgE, IgG1, and IgG2a in serum were measured in the mice with OVA-induced CARAS. OVA-specific IgE and OVA-specific IgG1 levels were notably elevated in the CARAS mice compared to the control group. However, FJE and Dex significantly controlled this upregulation ( Figure 4A,B). OVA-specific IgG2a in FJE 200 and Dex groups was significantly improved compared with the CARAS group ( Figure 4C).

FJE Suppressed Immunoglobulin Levels in Serum of the OVA-Induced CARAS Mouse Model
To investigate whether FJE affects allergic reactions, OVA-specific IgE, IgG1, and IgG2a in serum were measured in the mice with OVA-induced CARAS. OVA-specific IgE and OVA-specific IgG1 levels were notably elevated in the CARAS mice compared to the control group. However, FJE and Dex significantly controlled this upregulation ( Figure  4A,B). OVA-specific IgG2a in FJE 200 and Dex groups was significantly improved compared with the CARAS group ( Figure 4C).

FJE Regulated T Helper-Associated Cytokine Production in NLF and BALF of the OVA-Induced CARAS Mouse Model
The levels of Th2-associated cytokines IL-4, IL-5, IL-6, and IL-13 in both NLF and BALF were significantly up-regulated in the CARAS group compared with the control group. FJE 200 mg/kg and Dex were more efficient in suppressing these Th2-associated cytokines than the other treatments. However, the levels of IL-5 in the NLF were insignificant in all groups except FJE 200 mg/kg ( Figure 5D). Similarly, the levels of IL-4, IL-5, IL-6, and IL-13 were significantly higher in the BALF of the CARAS group, whereas FJE and Dex suppressed the levels of Th2-associated cytokines ( Figure 5). The levels of Th1-associated cytokines IL-12 and IFN-γ levels in both NLF and BALF were significantly lower in the CARAS group than in the control group. However, treatments with FJE and Dex improved IFN-γ and IL-12 levels in the NLF and BALF ( Figure 5A,B,H,I). Moreover, TNF-α, a pro-inflammatory cytokine, was also assessed to determine the effect of FJE on CARAS. The TNF-α levels in both NLF and BALF were significantly higher in the CARAS group than in the other treated groups. FJE dose-dependently down-regulated TNF-α, whereas levels were significantly lower in the FJE-treated groups than in the CARAS group.

FJE Regulates Epithelium-Derived Cytokines in NLF and BALF of the OVA-Induced CARAS Mouse Model
Epithelium-derived cytokines such as thymic stromal lymphopoietin (TSLP) and IL-33 play vital roles in linking innate and adaptive immune reactions associated with Th2 cytokine-mediated reactions [22,23]. TSLP and IL-33 are also potentially involved in the pathogenesis of asthma [23]. Hence, TSLP and IL-33 levels were measured to assess the effect of FJE on CARAS. TSLP was significantly higher in the CARAS group; however, FJE treatment significantly reduced these levels in both NLF and BALF. Notably, the administration of various concentrations of FJE resulted in similar effects on the TSLP level.
This suggests that the lowest concentration was adequate in regulating the TSLP level ( Figure 6A,C). However, in BALF, a dose-dependent regulation was observed. Furthermore, the level of IL-33 was significantly elevated in the NLF and BALF of the CARAS group. However, FJE and Dex significantly reduced these levels, to a greater degree than that in the CARAS group ( Figure 6B,D). Notably, the IL-33 level in the lung tissue clearly indicates the dose-dependent regulatory effects of FJE ( Figure 6E). Moreover, immunohistochemistry revealed significantly lower expression of ST2, the receptor for IL-33, in nasal tissues upon FJE and Dex treatment compared with levels in the CARAS group ( Figure 6F).

Induced CARAS Mouse Model
The levels of Th2-associated cytokines IL-4, IL-5, IL-6, and IL-13 in both NLF and BALF were significantly up-regulated in the CARAS group compared with the control group. FJE 200 mg/kg and Dex were more efficient in suppressing these Th2-associated cytokines than the other treatments. However, the levels of IL-5 in the NLF were insignificant in all groups except FJE 200 mg/kg ( Figure 5D). Similarly, the levels of IL-4, IL-5, IL-6, and IL-13 were significantly higher in the BALF of the CARAS group, whereas FJE and Dex suppressed the levels of Th2-associated cytokines ( Figure 5). The levels of Th1-associated cytokines IL-12 and IFN-γ levels in both NLF and BALF were significantly lower in the CARAS group than in the control group. However, treatments with FJE and Dex improved IFN-γ and IL-12 levels in the NLF and BALF ( Figure 5A,B,H,I). Moreover, TNF-α, a pro-inflammatory cytokine, was also assessed to determine the effect of FJE on CARAS. The TNF-α levels in both NLF and BALF were significantly higher in the CARAS group than in the other treated groups. FJE dose-dependently down-regulated TNF-α, whereas levels were significantly lower in the FJE-treated groups than in the CARAS group.

FJE Regulates the Inflammatory Response via the NF-κB Signaling Pathway in the OVA-Induced CARAS Mouse Model
Activation of NF-κB signaling is critical to the regulation of Th2 cytokine levels [24].
To determine the effect of FJE on mice with OVA-induced CARAS, NF-κB signaling was evaluated. The levels of P-NF-κB and P-IκB in NLF were significantly higher in the CARAS group than in the control group, while FJE down-regulated levels. Similar outcomes were observed in BALF ( Figure 7A-F). The NF-κB-related proteins, such as P-NF-κB and P-IκB, were noticeably expressed in the lung tissues of CARAS mice compared with control mice; in contrast, these were significantly reduced in FJE and Dex-treated mice ( Figure 7G).
suggests that the lowest concentration was adequate in regulating the TSLP level ( Figure  6A,C). However, in BALF, a dose-dependent regulation was observed. Furthermore, the level of IL-33 was significantly elevated in the NLF and BALF of the CARAS group. However, FJE and Dex significantly reduced these levels, to a greater degree than that in the CARAS group ( Figure 6B,D). Notably, the IL-33 level in the lung tissue clearly indicates the dose-dependent regulatory effects of FJE ( Figure 6E). Moreover, immunohistochemistry revealed significantly lower expression of ST2, the receptor for IL-33, in nasal tissues upon FJE and Dex treatment compared with levels in the CARAS group ( Figure 6F). Data represent mean ± SEM (n = 6/group). # p < 0.05, ## p < 0.01, ### p < 0.001 compared with control, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CARAS. Dex, Dexamethasone.

FJE Regulates the Inflammatory Response via the NF-κB Signaling Pathway in the OVA-Induced CARAS Mouse Model
Activation of NF-κB signaling is critical to the regulation of Th2 cytokine levels [24].
To determine the effect of FJE on mice with OVA-induced CARAS, NF-κB signaling was evaluated. The levels of P-NF-κB and P-IκB in NLF were significantly higher in the CA-RAS group than in the control group, while FJE down-regulated levels. Similar outcomes were observed in BALF ( Figure 7A-F). The NF-κB-related proteins, such as P-NF-κB and P-IκB, were noticeably expressed in the lung tissues of CARAS mice compared with control mice; in contrast, these were significantly reduced in FJE and Dex-treated mice ( Figure  7G).  and P-IκB in BALF. (G) Immunoblotting of NF-κB, P-NF-κB, IκB, and P-IκB in lung tissue and quantification of protein levels. Mice were administered saline, Dex (1.5 mg/kg), or FJE (50, 100, or 200 mg/kg) once a day for 16 days. Data represent mean ± SEM (n = 6/group). * p vs. CARAS and # p compared with control. # p < 0.05, ## p < 0.01, ### p < 0.001 compared with control, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CARAS. Dex, Dexamethasone.

Discussion
CARAS is a novel disorder linked to lower and upper lung inflammation. This combined inflammation immediately induces a coordinated response by mast cells and their mediators, influencing nasal congestion and vascular permeability. The use of polyphenolrich natural sources could reduce the potential repercussions associated with the coordinated response. Therefore, in this study, we examined the effect of polyphenol-rich FJE on CARAS and the associated mechanism in a mouse model. FJE inhibited symptoms, such as rubbing and sneezing, consistent with prior results in placebo-controlled trials showing that polyphenols relieved nose symptoms [25,26]. Further, the release of cytokines or inflammatory mediators causes the infiltration of inflammatory cells into the nasal mucosa, influencing the inflammatory response [27]. Airway inflammation is a crucial factor for the development of asthma and AR, as it causes morphological alterations in the airway via the infiltration of inflammatory cells such as neutrophils, eosinophils, and macrophages [28]. In this study, histopathological analysis revealed reduced mucosal edema, goblet cell hyperplasia, and a thinner subepithelial layer in the CARAS mouse model treated with FJE, suggesting reduced pathological injury. Consistent with previous reports [29], these findings demonstrate the efficacy of FJE in reducing OVA-induced inflammatory responses. Based on these results, we suggest that FJE exerts a protective effect on the OVA-induced CARAS mouse model. Allergic mouse models have established a significant association between antigenspecific IgE and IgG1 [30]. These immunoglobulins are markers for Th2 cells, whereas IgG2a is a marker for Th1 cells [31]. In this study, FJE suppressed OVA-specific IgE and IgG1 while stimulating IgG2a. Therefore, a higher IgG2a/IgG1 ratio is expected with FJE treatment, as a higher IgG2a/IgG1 ratio indicates a protective immune response in allergic inflammation diseases [32]. Furthermore, mast cell degranulation releases inflammatory mediators such as histamines, prostaglandins D2, kinins, heparin, and serine proteases [33]. In this study, the administration of FJE significantly inhibited mast cell degranulation, which may have contributed to decreased rubbing and sneezing in FJE-treated mice. In contrast, the control group exhibited the maximum degranulation rate, with increased rubbing and sneezing activities, because enhanced vascular permeability and localized edema block nasal airways and caused congestion [34]. These observations suggest that FJE exhibited an anti-allergic effect in the OVA-induced CARAS model.
Research into mitigating airway inflammation led to the discovery of critical roles for IL-33, Th2, Th1 cytokines, TNF-α, NF-κB, and TSLP in the development of allergic airway diseases. The imbalance between Th1 and Th2 cell-mediated immunity plays a crucial role in the pathophysiology of AR. Increased Th2 cell-secreted cytokines trigger B cells to generate allergen-specific IgE, which causes acute symptoms. IL-4 stimulates IgE synthesis, IL-5 is involved in eosinophilic growth and differentiation, and IL-13 is involved in mucus production and airway hyper-reactivity [35,36]. In this study, FJE administration reduced IL-4, IL-5, and IL-13 levels, which influenced IgE synthesis, mucus production, and eosinophilic growth and differentiation. Furthermore, Treg cells potentially promote and maintain allergy tolerance by modulating both the innate and adaptive immune responses triggered by allergens [37]. However, FOXP3, a transcriptional regulator of Treg cells, is key to suppressing Th2 responses upon exposure to allergens [38]. Treg cells exert an immunomodulatory effect by secreting anti-inflammatory cytokines such as IL-10, restricting the activity of different immune cells and ultimately reducing the immune responses. Here, FJF treatment significantly reduced Th2 cytokines such as IL-4, IL-5, and IL-13 and improved the IL-10 levels in BALF and lung homogenate ( Figure S1), suggesting improved Treg FOXP3. Similarly, previous investigations demonstrated that herbal extracts suppressed Th2 cytokines [39]. These reports strongly support the FJE study observations. Moreover, the downregulation of these cytokines was observed in both NLF and BALF after FJE treatment, confirming the influence of FJE. Moreover, the levels of Th2 cytokines such as IL-4 and IL-5 are significantly higher in patients with AR [40], which potentially inhibits the functions of the Th1 immune response [41]. Th1 cytokines, such as IFN-γ and IL-12, are essential for the development of allergic airway inflammation. IFN-γ and IL-12 are considered anti-inflammatory as they prevent allergies and manifestations of allergic inflammation [42]. Following these observations, FJE treatment stimulated IFN-γ and IL-12 levels in the OVA-induced CARAS mouse model. NF-κB is a transcription factor responsible for various immune and inflammatory responses [43]. The NF-κB signaling pathway plays a role in airway inflammation, and an elevated NF-κB level has been frequently observed in OVA-induced asthmatic mice. NF-κB contributes to cytokine production and plays a vital role in the development of inflammatory diseases [44][45][46]. Additionally, NF-κB is involved in T cell differentiation, Th0 differentiation, and IL-4 induction, affecting the differentiation of naive T cells and mediating the induction of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α. These pro-inflammatory cytokines then activate NF-κB [47]. Thus, we examined NF-κB and P-NF-κB and P-IκB, components of the NF-κB signaling pathway, to explore their involvement in the FJE-induced protective effect. We found that FJE efficiently controlled the expression of P-NF-κB and P-IκB, suggesting an influence on cytokine production. Our observations suggest that FJE is approximately as effective as Dex at inhibiting airway inflammation.
TSLP, a pleiotropic cytokine, is a key factor in the pathogenesis of asthma, and elevated TSLP levels have been correlated with enhanced levels of TNF-α and Th2 cytokines [48]. TSLP is produced by epithelial cells and is triggered by various environmental stimuli; it plays a critical role in producing Th2 cytokines such as IL-33 and IL-25 and inducing Th2-type differentiation of CD4+ T cells. These associations influence the adaptive immune responses affecting respiratory disorders [24]. Additionally, TSLP is regulated by inflammatory mediators and TNF-α in an NF-κB-dependent manner [49]. In our study, reduced levels of TSLP were observed and correlated with lower levels of TNF-α and Th2 cytokines. These events may be involved in the underlying molecular mechanism of mitigating respiratory disorders by FJE. Additionally, ST2 is linked to asthma susceptibility, and recent investigations have shown that epithelial cells, endothelial cells, mast cells, and airway smooth muscle cells express the ST2 receptor [50,51]. IL-33 binding to ST2 recruits IRAK, IRAK4, MyD88, and TRAF6. ST2 activates NF-κB and MAP kinases, triggering pro-inflammatory effects [50,52]. Thus, inhibiting ST2-mediated inflammation may reduce the aggravation of airway inflammation. We found that administration of FJE significantly lowered ST2 expression. These observations confirm the potential effectiveness of FJE on CARAS. Moreover, all these positive implications of FJE can be attributed to the presence of EMODIN glucoside 1, EMODIN glucoside 2, procyanidin, and other polyphenols. The ultra-performance liquid chromatography data indicate that FJE contains a substantial quantity of these biologically active compounds ( Figure S2). These compounds are known to suppress inflammation by inhibiting leukocyte movement, preventing β-cell destruction and alleviating airway inflammation [20,[53][54][55]. In addition, procyanidins assist in prevention of airway inflammation [56][57][58]. Together, resveratrol's potent anti-inflammatory and antioxidant properties and EMODIN's anti-inflammatory properties help to reduce airway inflammation. In addition, Procyanidin's potential to improve vascular health indirectly contributes to optimal oxygen transport and minimizing inflammation-associated damage [59][60][61]. Hence, the presence of these biologically active compounds positively influences multiple signaling pathways and regulates airway inflammation-associated disorders.
CARAS is a unified airway disease characterized by lower and upper lung inflammation with AR and asthma. The symptoms include airway hyper-responsiveness, mucus hypersecretion, and eosinophilic infiltration in the airways. Our data indicate that FJE exerts a protective effect against OVA-induced CARAS in mice by reducing the infiltration of inflammatory cells, regulating the accumulation of mucus, suppressing OVA-specific immunoglobulins, and modulating the IL-33/TSLP/NF-κB signaling pathway (Figure 8). The major observations of this study are consistent with those of pathways observed in human clinical trials. Future studies on cytokines such as IL-17 and the involvement of immune cells such as B cells, lymphoid, and NKT cells are required.

Preparation of Fallopia Japonica Root Extract
FJE was provided by the Natural F&P Co., Ltd. (Cheongju-si, Republic of Korea). Briefly, F. japonica plants were harvested from Jecheon-si (Chungcheong-do, Republic of Korea) and Youngcheon (Gangwon, Republic of Korea). Raw F. japonica was dried, pulverized, and extracted with 10 times the volume of distilled water by heating (±50 °C) for 6 h. FJE was filtered using a cartridge filter and concentrated using a Rotavapor R-210 (BÜCHI Labortechnik AG, Flawil, Switzerland) at 50 °C in a vacuum, followed by drying.

Animal Studies
Five week old male BALB/c mice were obtained from the Damool Science Co. (Daejeon, Korea). Animals were housed under pathogen-free conditions with a 12 h:12 h light/dark cycle with free access to food and water. The animal experiments were carried out following the Jeonbuk National University Animal Care and Use Committee guidelines (CBNU 2021-0115).

CARAS Model and Treatment
Animals were acclimatized for 1 week before use. Six-week-old mice were randomly segregated into 6 groups, with 6 mice in each group. Group 1 was the control group, which was sensitized, treated, and challenged with saline; Group 2 was the CARAS group, in which mice were sensitized and challenged with OVA; Groups 3-5 were the FJE groups, in which CARAS mice were treated with 50, 100, or 200 mg/kg of FJE; and Group 6 was Figure 8. The molecular mechanism underlying the beneficial effects of FJE in the OVA-induced CARAS mouse model. Exposure to allergens induces the release of IL-33 and TSLP from epithelial cells and promotes the differentiation of Th0 cells to Th2. This stimulates B cells to produce IgE, which binds and activates mast cells. Activated mast cells trigger the release of pro-inflammatory cytokines and chemokines. Together with Th2 cell-released cytokines, leukocytes are recruited and activated, leading to allergic inflammatory symptoms. Moreover, the NF-κB pathway is activated to amplify the inflammatory condition. FJE exhibited anti-allergic inflammatory effects via the IL-33/TSLP/NF-κB signaling pathway.

Preparation of Fallopia Japonica Root Extract
FJE was provided by the Natural F&P Co., Ltd. (Cheongju-si, Republic of Korea). Briefly, F. japonica plants were harvested from Jecheon-si (Chungcheong-do, Republic of Korea) and Youngcheon (Gangwon, Republic of Korea). Raw F. japonica was dried, pulverized, and extracted with 10 times the volume of distilled water by heating (±50 • C) for 6 h. FJE was filtered using a cartridge filter and concentrated using a Rotavapor R-210 (BÜCHI Labortechnik AG, Flawil, Switzerland) at 50 • C in a vacuum, followed by drying.

Animal Studies
Five week old male BALB/c mice were obtained from the Damool Science Co. (Daejeon, Korea). Animals were housed under pathogen-free conditions with a 12 h:12 h light/dark cycle with free access to food and water. The animal experiments were carried out following the Jeonbuk National University Animal Care and Use Committee guidelines (CBNU 2021-0115).

CARAS Model and Treatment
Animals were acclimatized for 1 week before use. Six-week-old mice were randomly segregated into 6 groups, with 6 mice in each group. Group 1 was the control group, which was sensitized, treated, and challenged with saline; Group 2 was the CARAS group, in which mice were sensitized and challenged with OVA; Groups 3-5 were the FJE groups, in which CARAS mice were treated with 50, 100, or 200 mg/kg of FJE; and Group 6 was the Dex group, in which CARAS mice received 1.

Collection of Serum, NLF, and BALF
Mice were anaesthetized with diethyl ether 24 h after the last challenge, and blood samples were collected from eye sockets. The collected blood was centrifuged at 10,000 rpm/min at 4 °C to obtain serum. NLF was collected as described previously using an 18gauge catheter. Briefly, the trachea was partly eviscerated; the catheter was inserted to perfuse phosphate-buffered saline (PBS), and the collected NLF was centrifuged. The supernatant was used for ELISA, the cell pellet was suspended in PBS, and total cell numbers were counted using a cytospin (TH-CYTO4, Thermo Electron Corporation, Waltham, MA, USA, Diff-Quik staining (38,721, Sysmex, Chuo-ku, Kobe, Hyogo, Japan), which was performed following the manufacturer's instructions.

Collection of Serum, NLF, and BALF
Mice were anaesthetized with diethyl ether 24 h after the last challenge, and blood samples were collected from eye sockets. The collected blood was centrifuged at 10,000 rpm/min at 4 • C to obtain serum. NLF was collected as described previously using an 18-gauge catheter. Briefly, the trachea was partly eviscerated; the catheter was inserted to perfuse phosphate-buffered saline (PBS), and the collected NLF was centrifuged. The supernatant was used for ELISA, the cell pellet was suspended in PBS, and total cell numbers were counted using a cytospin (TH-CYTO4, Thermo Electron Corporation, Waltham, MA, USA, Diff-Quik staining (38,721, Sysmex, Chuo-ku, Kobe, Hyogo, Japan), which was performed following the manufacturer's instructions.

Immunohistochemistry
Immunohistochemistry was performed using a Rabbit specific HRP/DAB (ABC) Kit (ab64261, Abcam, Cambridge, UK) and anti-ST2 antibody (MBS7604494, MyBioSource, San Diego, CA, USA). Briefly, the sections were brought to boiling temperature in 1× citrate buffer (ab93678, Abcam, Cambridge, UK), pH 6.0, and maintained at a sub-boiling temperature for 10 min for antigen retrieval. Sections were incubated with a blocking solution and incubated overnight at 4 • C with an anti-ST2 antibody (1:500). The sections were subsequently incubated with a biotinylated goat anti-rabbit IgG secondary antibody. Staining was developed using DAB solution for 1 min (1:50; Millipore, Billerica, MA, USA). The ST2-positive area was measured using the Fiji application.

Western Blot Anlaysis
Collected lung tissues were homogenized and lysed with RIPA buffer. Protein samples (50 µg) were separated by 10% SDS-PAGE and transferred onto a PVDF membrane, which was then blocked with 5% skimmed milk at room temperature for 1 h and incubated with primary antibodies against NF-κB

Statistical Analysis
All the statistical analyses were performed with GraphPad Prism version 8.4 (Graph-Pad Software, Boston, MA, USA). Data are expressed as mean ± SEM. Student's t-test and one-way ANOVA with Dunnett's test were used to determine group differences. The significance level was set at p < 0.05.

Conclusions
Our findings provide substantial evidence for the anti-asthmatic and anti-inflammatory effects of FJE on an OVA-induced CARAS mouse model. FJE attenuated airway inflammation by suppressing inflammatory cells and decreasing serum anti-OVA IgE, anti-OVA IgG1 and pro-inflammatory cytokines in mice with OVA-induced CARAS. At the molecular level, FJE ameliorated CARAS by modulating the IL-33/TSLP/NF-κB signaling pathway. These findings suggest that FJE is a promising therapeutic functional food for airway disorders.

Data Availability Statement:
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.